Growth regulation in the mammary gland in tissue culture involves at least three factors - the type of mammary epithelium under investigation, i.e. whether isolated from a pregnant, non-pregnant or lactating animal; the hormones used, either alone or in combination and the interaction of epithelial cells with tissue culture substrates. We are particularly interested in the importance of epithelial interactions with cellular and non-cellular substrates that modify growth and differentiation. The principal cellular interaction of epithelial cells within the mammary gland during hormone mediated growth and differentiation is with adipocytes. We have developed an in vitro model system to test the hypothesis that interaction with adipocytes influences mammary epithelial growth and milk protein synthesis. Since adipocytes float and do not attach in cell culture, we have taken advantage of the 3T3-L1 cell line which initially attaches to culture dishes before the cells differentiate into adipocytes. The 3T3-L1 adipocytes have physiological and biochemical properties found in adipocytes. Thus, the importance of direct adipocyte-epithelial cell interactions in promoting cell growth, response to mitogens, and hormonal regulation of milk protein (casein) synthesis, will be tested by plating mammary epithelium directly on adipocyte cell layers. Indirect actions mediated through extracellular components produced by adipocytes (extracellular matrix, substrate attached material) will be studied by using culture dishes upon which adipocytes have previously grown, or culture medium previously conditioned by adipocytes. Preliminary experiments demonstrate that adipocytes and extracellular components produced by adipocytes greatly enhance the growth of mammary epithelial cells.